本文選題：乙肝病毒　切入點：納米技術　出處：《首都醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:1.建立納米磁珠提取血清乙型肝炎病毒脫氧核糖核酸(hepatitis B virus deoxyribonucleic acid,HBV DNA)聯(lián)合量子點標記熒光探針檢測HBV變異位點(M204I位點突變)的新方法。2.探討新方法檢測M204I位點突變在評價慢性乙型肝炎(chronic hepatitis B,CHB)核苷(酸)類(nucleos(t)ide analogues,NAs)藥物治療應答不佳患者中的意義。方法:將羅氏COBAS Taq Man檢測的病毒載量為107IU/m L的血清樣本,倍比稀釋成不同濃度的HBV DNA作為血清標準品(理論值),分別用本方法(試劑1)、磁珠法(試劑2)、煮沸法(試劑3)三種方法提取HBV DNA,從檢測結(jié)果與理論值的相關性以及可重復性方面比較本實驗的納米磁珠提取方法與國產(chǎn)磁珠法核酸自動提取法和傳統(tǒng)煮沸法的提取效果。收集臨床符合耐藥,且直接測序法檢測到有M204I位點突變的血清樣本8例,不含有M204I位點突變的血清樣本15例,設計并確定含有M204I位點突變的引物及探針序列,用量子點標記熒光探針雜交法檢測HBV M204I變異位點,與直接測序法的檢測結(jié)果進行比較,評價新方法檢測位點變異的診斷效能,以及此方法在檢測核苷(酸)類藥物治療應答不佳的慢乙肝患者HBV M204I位點變異時的意義。結(jié)果:納米磁珠提取HBV DNA方法與理論值的相關性:試劑1、試劑2、試劑3提取HBV DNA定量與理論值相關性分析的r值分別為0.986(P0.001)、0.950(p=0.001)、0.979(P0.001),差異均具有統(tǒng)計學意義。三種方法檢測的相對偏差均值分別為0.243±0.405(試劑1)、1.189±0.855(試劑2)、-0.439±0.618(試劑3)。在檢測低水平HBV DNA定量時,理論值為101IU/ml,試劑1為3.520×101IU/ml,試劑2為9.123×103IU/ml,試劑3為6.195×101IU/ml。可重復性:試劑1對同一樣本在不同時間檢測結(jié)果的相對偏差均值為0.505±0.659。量子點標記熒光探針檢測HBV M204I變異位點的檢測下限是103IU/ml,且病毒水平越高,熒光光點越密集。利用量子點標記熒光探針法對臨床中出現(xiàn)對核苷(酸)類藥物治療應答不佳的28例慢乙肝患者的HBV M204I位點進行檢測,并與直接測序法比較,其中11例測序陽性樣本中均檢測到M204I位點的突變,17例測序陰性樣本中均未檢測到M204I位點的突變,兩種方法檢測結(jié)果一致。結(jié)論:利用納米磁珠提取HBV DNA聯(lián)合量子點探針雜交法檢測病毒HBV DNA聚合酶基因變異的方法靈敏、準確,簡便易行,為檢測乙型肝炎病毒M204I位點變異提供了一個新的手段,且HBV DNA聚合酶位點變異可能是導致慢乙肝患者抗病毒治療過程中發(fā)生應答不佳的原因之一。
[Abstract]:Objective 1. To establish a new method for the detection of mutation of HBV mutation site M204I by using nanomagnetic beads to extract hepatitis B virus deoxyribonucleic deoxyribonucleic DNA from serum. 2. To explore a new method for detection of M204I locus. The significance of mutagenesis in the evaluation of chronic hepatitis B hepatitis nucleostidine nucleostidine (analoguestide) in patients with poor response to drugs. Methods: serum samples with a viral load of 107 hepatitis / mL detected by COBAS Taq Man were used to evaluate the efficacy of the mutation in the treatment of chronic hepatitis B patients with adverse drug response. HBV DNA of different concentration was diluted into different times ratio as the standard serum (theoretical value). The method was used to extract HBV by three methods (reagent 1n, magnetic bead method (reagent 2N), boiling method (reagent 3)). The correlation between the results of detection and the theoretical value was analyzed. The results of extraction of nanomagnetic beads were compared with those of domestic magnetic beads and traditional boiling methods. The clinical results were consistent with drug resistance. In addition, 8 serum samples with M204I mutation and 15 serum samples without M204I mutation were detected by direct sequencing. Primers and probe sequences containing M204I mutation were designed and determined. The mutation sites of HBV M204I were detected by quantum dot labeled fluorescence probe hybridization and compared with the results of direct sequencing to evaluate the diagnostic effectiveness of the new method. And the significance of this method in detecting the variation of HBV M204I site in patients with chronic hepatitis B who have poor response to nucleoside (acid) drugs. Results: the correlation between the method of HBV DNA extraction by nanomagnetic beads and the theoretical value is as follows: reagent 1, reagent 2, reagent 3. The r values of quantitative and theoretical analysis of HBV DNA were 0.986p 0.001 ~ 0.950p 0.001C ~ (0.979) P 0.001 ~ (-1), respectively. The mean relative deviations of the three methods were 0.243 鹵0.405 (reagents 1.189 鹵0.855) (reagent 2-0.439 鹵0.618) (reagents 2-0.439 鹵0.618). The relative deviations of the three methods were 0.243 鹵0.405 (reagent 2-0.439 鹵0.618) and 0.43 鹵0.405 (reagents 2-0.439 鹵0.618), respectively. The theoretical value is 101 IUU / ml, reagent 1 is 3.520 脳 10 ~ (-1) IUP / ml, reagent 2 is 9.123 脳 10 ~ (3) IUP / ml, reagent 3 is 6.195 脳 10 ~ (-1) IUP / ml. Repeatability: the average relative deviation of reagent 1 to the same sample at different times is 0.505 鹵0.659. The detection of HBV M204I variation site by fluorescence probe labeled by quantum dot is 0.505 鹵0.659. The lower limit is 103 IU / ml, and the higher the virus level, The denser the fluorescent spot was, the more intense the fluorescent spot was. The HBV M204I loci in 28 patients with chronic hepatitis B who had a poor response to nucleoside (acid) drugs were detected by quantum dot labeling fluorescence probe method, and compared with the direct sequencing method. M204I locus mutations were detected in 11 sequencing positive samples and no M204I mutation was detected in 17 sequencing negative samples. Conclusion: the method of HBV DNA extraction and quantum dot hybridization is sensitive, accurate and easy to detect the mutation of virus HBV DNA polymerase gene. It provides a new method to detect the mutation of M204I site of hepatitis B virus, and the mutation of HBV DNA polymerase site may be one of the causes of poor response to antiviral therapy in patients with chronic hepatitis B.
【學位授予單位】：首都醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62

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